Daniel:Notebook/HiResChrPaint/2014-1-14: Difference between revisions

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>Djacobse
>Djacobse
 
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*Human Cot-1 DNA, 1 ug
*Human Cot-1 DNA, 1 ug


  0. Vaccuum centrifuge DNA; resuspend in 2.5 uL nfH2O
  0. Vaccuum centrifuge DNA; resuspend in 6 uL nfH2O +3 uL sodium bicarbonate buffer
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 1.5 uL of sodium bicarbonate buffer
  2. Add 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  3. Dissolve 1 vial of reactive dye in 4 uL solvent; vortex ≥10 seconds
4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
     *dye cannot be saved for later use.  Use immediately!
     *dye cannot be saved for later use.  Use immediately!
  5. Add 1 uL dye to sample
  4. Add 1 uL dye to each sample
  6. Incubate in the dark for 1 hour
  5. Incubate in the dark for 1 hour
  7. Add 20 uL nfH20 to sample; Measure concentration in Nanodrop
  6. Add 20 uL nfH20 to sample;
7. Take 5 uL from sample and dilute with 45 uL nfH2O; Measure concentration in Nanodrop
  8. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]]
  8. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]]
  9. Measure concentration in Nanodrop
  9. Measure concentration in Nanodrop

Latest revision as of 21:45, 15 January 2014

Alu Control (Started 1/08/2014)[edit]

Back to Calendar

Dye Coupling Test (with Positive Control)[edit]

Repeating the dye coupling test one last time using a positive control as well.

Samples

  • V6S3-A; 13.5 uL, total 1.0 ug
  • Alu2-D; total 1.0 ug
  • Human Cot-1 DNA, 1 ug
0. Vaccuum centrifuge DNA; resuspend in 6 uL nfH2O +3 uL sodium bicarbonate buffer
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Add 3 uL of sodium bicarbonate buffer
3. Dissolve 1 vial of reactive dye in 4 uL solvent; vortex ≥10 seconds
   *dye cannot be saved for later use.  Use immediately!
4. Add 1 uL dye to each sample
5. Incubate in the dark for 1 hour
6. Add 20 uL nfH20 to sample; 
7. Take 5 uL from sample and dilute with 45 uL nfH2O; Measure concentration in Nanodrop
8. Centri-Sep column purification
9. Measure concentration in Nanodrop

Labeling Results[edit]

Sample ng/uL dsDNA pmol/uL dye pmol/uL ds probe dye/100bp ng/uL ssDNA pmol/uL dye pmol/uL ss probe dye/100bp
Alu2D-Pre Column 170 115     113.2 116.6    
Blank-Pre Column 157.4 111     103.3 110.6    
Cot1-Pre Column 150.9 105     102.1 106.4    
V6S3-Pre Column 160.3 109.2     109.6 112.9    
Alu2D-Post Column 24.4 0.5 0.4 1.4 12.6 0.5 0.4 1.3
Blank-Post Column 1 0 0.0 0.0 0.2 0 0.0 0.0
Cot1-Post Column 25.9 1.7 0.2 4.3 15.6 1.3 0.3 2.8
V6S3-Post Column 41.3 0.6 0.8 1.0 24.4 0.3 0.9 0.4

This is a big table, but there are a couple of messages to take away from this.

  • Excess dye causes a false DNA reading. It doesn't appear to have an effect when dye concentration is low, but when the dye concentration was high (pre-spin column), even the blank sample measured a considerable amount of DNA. I'd recommend always using a blank column and subtracting the background from future samples if pre-purification measurements are needed.
  • The Cot1 DNA is reproducibly eluting with dye. Given the other labeling results from previous days (1/13/2014 and 1/10/2014), I'd say that the Cot1 DNA, although having no amine groups, might be being labeled anyway. Or, at least, it's clogging up the column and allowing the dye to co-elute.